We identified a number of cell lines which can be transfected with RNA transcribed in vitro from our infectious cDNA clone and showed that the genomes replicate and that infectious virus is produced. HEV replicons expressing green fluorescent protein or luciferase were used to compare the effects of mutations in a non-coding region on replication in cell culture compared to in rhesus macaques. We developed a Hep G2 cell culture system which permits limited infection by wild-type HEV and used it to study neutralization and to show that HEV is more heat labile than is hepatitis A virus. We isolated subclones of Huh7 cells that differed in infectability by HEV and showed that HEV produced in transfected cells could infect the best of these subclones. We used these cells to confirm that monoclonal antibodies deemed "neutralizing" based on indirect tests, did indeed neutralize virus produced in vivo or in vitro. We found that HEV replication involves at least one subgenomic RNA and we mapped its 5' terminus. We studied the humoral immune response to the HEV recombinant vaccine we developed previously and mapped the neutralization site, demonstrated that the vaccine induces antibodies that cross-react with viruses from all four genotypes, and showed that the vaccine contains all the immunodominant epitopes located in the capsid protein.